Keratinization is an orderly process in which protein-containing monomers are synthesized and assembled in viable epidermal cells, keratinocytes. The final product, keratin, is not secreted but is retained within the cellular envelope in non-viable anucleate cells at the skin surface. Recently, covalent cross-links were identified in wool keratin and shown to be epsilon-gamma-glutamyl lysine bonds. This observation may, in part, explain the durability of keratin and its resistance to solubilization and biochemical analysis. Transglutaminase, an enzyme which catalyzes the formation of gamma-glutamyl-epsilon-lysine bonds, has been isolated in soluble form and purified from guinea pig hair follicles and bovine and rat epidermis (Buxman and Wuepper, in preparation). We propose to analyze epidermal transglutaminase (ET) in the skin of experimental animals (cow and rat) and man. ET will be purified by chromatographic and electrophoretic methods and assayed by its ability to incorporate the fluorescent substrate, dansyl cadaverine, into casein. Keratinocyte transglutaminase will be strictly compared to and distinguished from plasma transglutaminase (fibrin stabilizing factor, factor III) by enzymatic, physical and immunochemical properties. The enzyme ET will be localized in epidermis and epidermal appendages by histochemical or immunochemical methods by light-or electron-microscopy. ET will be employed as a biochemical probe to identify native soluble donor and acceptor proteins in epidermis. In order to assess the functional importance of the enzyme, ET will be employed as a biochemical probe to identify native soluble donor and acceptor proteins in epidermis. In order to assess the functional importance of the enzyme, ET will be inhibited in cultures of skin in vitro. Finally, ET will be measured in human diseases of keratinization, ectodermal dysplasias, or disorders of epidermal cell adhesion.